The utility of monoclonal antibodies extends from basic research to therapeutic and diagnostic applications. The ability to conjugate antibodies to functional agents extends their functionality even further. The manufacture of conjugated antibodies usually involves conjugation of a linker, drug, or other functional agent to reactive lysine or cysteine residues on the heavy (HC) and light (LC) chains of a monoclonal antibody (mAb). Lysine conjugation is typically mediated by succinimide (NHS)-based or isothiocyanate-based chemistry. Given the number of exposed lysines on the surface of an antibody, amine-based conjugation approaches result in multiple lysines being modified, though not all lysine residues are modified to the same extent. Therefore, the final product is a heterogeneous mixture of mAbs with a distribution of drug-to-antibody (DAR) ratios.
Most cysteines within an antibody are involved in either inter- or intra-chain disulfide bonds. Conjugation to cysteines thus requires at least partial reduction of the antibody. Like lysine-based conjugation, cysteine-based conjugation results in a heterogeneous mixture of conjugated antibodies differing in drug load and conjugation site. Each species of conjugated antibody may have distinct properties, which in turn could lead to wide variation of in vivo pharmacokinetic properties. Additionally, such heterogeneity can present challenges in manufacturing of the conjugated antibody.